1. Field of the Invention
The present invention relates to immunochemical assays. More particularly, the present invention relates to enzyme-linked immunosorbent assay (ELISA) for detecting, in a sample of human body fluid, the presence of antibodies having binding affinity for (1) Staphylococcus aureus REPB 275-290 or other homologous bacterial REPB proteins and (2) Arg-Lys-Leu-Lys (SEQ ID NO: 1) bacterial or viral proteins that mimic the N-terminal and C-terminal human opioid peptide dynorphin A 1-17, respectively.
2. Description of Related Art
An immune response can be specific for restricted sequences of immunogenic proteins (epitopes) as small as four amino acids in length. Identical epitopes can be present on proteins from organisms as different as plants, bacteria, viruses, parasites, fungi, and humans. Thus, by chance, unrelated proteins may display areas of sequence or conformational homology (molecular mimicry). Repeated exposure to the same epitope on disparate and seemingly inconsequential environmental antigens can result in sensitization to, and loss of immune tolerance for, similar epitopes on host proteins. Eventually the immune response to the epitope may precipitate disease. Because the epitope is the target, the organism that initiated the cascade need no longer be present for the development of disease. If there is significant immunogenicity and cross-reactivity, the immune response to the environmental epitope may become misdirected against its human counterpart.
Molecular mimicry is important to the development of epitope-specific autoimmunity. For example, a six amino acid peptide shared by myelin basic protein and hepatitis virus polymerase causes experimental autoimmune encephalomyelitis in rabbits. This correlates with the development of clinical autoimmune encephalitis for myelin basic protein in humans after immunization with Semple vaccine, a preparation of rabies-infected animal tissue. A sequence shared by coxsackie B3 virus and myosin causes myocarditis in mice. This fits observations of clinical myocarditis in humans following infection with coxsackie virus.
Alterations in serum immunoglobulins and proteins associated with inflammation, and elevated antiviral titers point to an active infectious or autoimmune etiology of psychiatric disorders, but are not always demonstrated. Epidemiological surveys to link specific disorders to specific viral infections are provocative, but nonconclusive.
Despite this finding, psychiatric disorders have clinical and epidemiological association with infection. Moreover, infections characterized by consistent psychiatric sequelae exhibit molecular mimicry of host proteins, cross-reactive autoimmunity (i.e., epitope-specific autoimmunity), induction of idiotypic networks, and, in some cases, alterations of central neurotransmitters. This raises the consideration that the psychiatric sequelae might be a consequence of these factors.
Streptococcal infection is a prototypic cause of epitope-specific autoimmunity for heart, kidney, and brain. Disease may occur despite elimination of Streptococcus pyogenes from the body. Streptococcal infections are also associated with psychiatric sequelae. Prior to effective treatment for rheumatic fever schizophrenic-like symptoms appeared either during acute illness or many years after resolution of the disease. This led to hypotheses using rheumatic fever as a paradigm for an infectious origin of schizophrenia.
Despite modern treatment, rheumatic fever continues to present an array of psychiatric sequelae. Obsessive-compulsive disorder (OCD) occurs with Sydenham's chorea. Sydenham's chorea is a neurological disorder seen in 20 percent of the cases of rheumatic fever. Sydenham's chorea is a consequence of an autoimmune response for basal ganglia proteins that cross-react with Streptococci. Clinical obsessiveness is noted in half the cases of Sydenham's chorea. This exceeds the 0.4 percent prevalence for OCD in adolescence. Outbreaks of rheumatic fever in the midwest and Pennsylvania were characterized by a high incidence of OCD with significant obsessional scores on the Yale-Brown Obsessional Inventory and the Leyton Obsessional Inventory.
Like Sydenham's chorea primary OCD is linked to abnormalities of the basal ganglia. Furthermore, OCD shows genetic linkage to Gilles de la Tourette's syndrome, a basal ganglia disorder associated with obsessive symptoms.
The principal neuropeptides of the basal ganglia include dynorphins. Opiates have regulatory influence on serotonergic nuclei of the median raphe which send inhibitory projections to the basal ganglia. The possibility that dynorphin may be altered in OCD is raised by depletion of dynorphin in the striatum of patients with Gilles de la Tourette's syndrome.
There are three main branches of opiate peptides. Pro-opiomelanocortin, a pituitary peptide, is the parent peptide for beta-endorphin, corticotropin (ACTH), and alpha-melanocortin (MSH). Proenkephalin is present in both the adrenal gland and brain and is the precursor for methionine and leucine enkephalins, while prodynorphin is present in cortex and is the precursor for dynorphin peptides and neo-endorphins.
Opioid peptides are ubiquitous antigens produced by vegetables, parasites, bacteria, and vertebrates. As a result, sensitization to opioid peptides as environmental antigens might induce cross-reactive immunity for endogenous opioid peptides. Alterations in opioid peptides are implicated in the pathogenesis of a number of psychiatric disorders. There is the chance that epitope-specific autoimmunity for these peptides may contribute to these alterations.
Some patients with major depressive disorder (MDD) and OCD produce antibodies for dynorphin. In a study of 105 subjects, OCD and MDD (clinically and biologically related disorders) showed greater reactivity for prodynorphin 209-240 than healthy volunteers, schizophrenics, Alzheimer's disease, multiple sclerosis, and AIDS patients as illustrated in FIG. 1. The absence of reactivity in AIDS and MS illustrates that the antibodies are not a nonspecific consequence of inflammation or infection. The absence of antibodies in schizophrenia and Alzheimer's disease suggests that the antibodies are not nonspecifically related to OCD and MDD. Comparison with age-matched volunteers is shown in FIG. 2.
Antidynorphin antibodies have an unknown origin. However, since some bacteria, plants, and parasites produce peptides with opiate-like immunoreactivity, the antibodies may occur in response to opioid-like epitopes of microbial proteins. If this were true then antidynorphin antibodies might cross-react on synthetic opioid-like sequences from microbial proteins.
Replication B protein 275-290 from Staphylococcus aureus plasmids (REPB) has homology with the amino acid sequence for dynorphin A 1-16 (Table 1).
TABLE 1 __________________________________________________________________________ Homology Between Dynorphin A and Bacterial Replication Protein B 275-290 Peptide Amino Acid Sequence __________________________________________________________________________ Dynorphin A 1-16 (SEQ ID NO:2) REPB 275-290 (SEQ ID NO:3) ##STR1## __________________________________________________________________________ Identical residues are represented by (.linevert split.) and analogous residues are represented by (:). The sequence REPB 275-290 is identical across Staphylococcus aureus, Lactobacillus plantarum and Bacillus subtilis, has 81 percent similarity and 50 percent identity with Dyn A 1-16. The greater similarity is for the enkephalin moiety, Tyr--Gly--Gly--Phe--Leu. (SEQ ID NO: 4).
The greater similarity is between the leucine-enkephalin moiety of dynorphin A (Tyr-Gly-Gly-Phe-Leu) (SEQ ID NO: 4) and REPB 275-279 (SEQ ID NO: 5) (Tyr-Gly-Gly-Leu-Leu.sup.5). REPB epitopes that are identical to S. aureus REPB 275-290 are present on Lactobacilli plantarum and Bacillus subtilis.
Bacterial plasmids are extrachromosomal DNA that determine important proteins. REP proteins are products of related plasmids present in Staphylococcus, Bacillus, Lactobacilli, Streptococci, Streptomyces, and Clostridia species. These organisms cause common infections and colonize anatomic sites (conjunctiva, skin, oral cavity) with intense sensory innervation. This allows access to sensory ganglia which have no blood brain barrier and direct projections to areas of the central nervous system that have high concentrations of dynorphin.
Streptococcus mutants and Lactobacilli colonize the oral cavity and cooperate to cause dental caries. Streptococcus mutants erodes enamel and promotes secondary invasion of dentine by Lactobacilli. Dental caries are highly prevalent infections and afford access to the central nervous system along the trigeminal nerves. As part of the normal oral flora these organisms may place susceptible humans at risk to the development of antibodies for dynorphin as a consequence of molecular mimicry.
A second set of proteins with homology to dynorphin A derive from a search for proteins with similarity to Arg-Lys-Leu-Lys, (SEQ ID NO: 1), a sequence that has similarity to dynorphin Arg-Pro-Lys-Leu-Lys.sup.13 (SEQ ID NO: 6), after introduction of a gap for maximum alignment. Arg-Lys-Leu-Lys (SEQ ID NO: 1) is a widely distributed epitope, present on both environmental and human proteins (Table 2).
TABLE 2 ______________________________________ Proteins that Display Arg--Lys--Leu--Lys Peptide Sequence Amino Acid ______________________________________ Microorganisms: Streptococcus pyogenes 6M -27 to -30 Arg--Lys--Leu--Lys. (SEQ ID NO: 1) Staphylococcus aureus exfoliative toxin B Arg--Lys--Leu--Lys 40--43 (SEQ ID NO: 1) Plasmodium falciparum calmodulin Arg--Lys--Leu--Lys 241-244 (SEQ ID NO: 1) Influenza B hemagglutinin 2 481-484 Arg--Lys--Leu--Lys (SEQ ID NO: 1) E. coli phosphoenolpyruvate carboxylase Arg--Lys--Leu--Lys 109-112 (SEQ ID NO: 1) Saccharomyces cerevisiae (Baker's yeast) Arg--Lys--Leu--Lys fatty acid synthase 412-415 (SEQ ID NO: 1) Human Proteins: GABA receptor (SEQ ID NO: 7) Arg--Lys--Ala--Lys Glutamate decarboxylase 308-311 Arg--Lys--Leu--Lys (SEQ ID NO: 1) Rabbit calsequestrin 259-262 Arg--Lys--Leu--Lys (SEQ ID NO: 1) Ca.sup.++ /calmodulin-dependent protein Arg--Lys--Leu--Lys kinase 298-301 (SEQ ID NO: 1) Ryanodine (calcium-channel) receptor Leu--Lys--Leu--Lys 4059-4062 (SEQ ID NO: 8) Human DNA polymerase 1431-1434 Arg--Lys--Leu--Lys (SEQ ID NO: 1) Gibbon IL-3 128-131 Arg--Lys--Leu--Lys (SEQ ID NO: 1) Diffuse B cell lymphoma proto-oncogene Arg--Lys--Leu--Lys 169-172 (SEQ ID NO: 1) ______________________________________
In particular it is present on several Streptococcus pyogenes and S. mutants proteins, Influenza B virus hemagglutinin, 472-475, and Plasmodium falciparum calmodulin. These organisms are associated with post-infectious neuropsychiatric disorders and autoimmune phenomena secondary to molecular mimicry. Staphylococcus aureus exfoliative toxin B (impetigo), Saccharomyces cerevisiae (Baker's yeast), Bacillus subtilis, Escherichia coli, and Epstein-Barr virus (infectious mononucleosis) proteins also display Arg-Lys-Leu-Lys (SEQ ID NO: 1).
These organisms are common causes of disease, and in the case of Baker's yeast and B. subtilis, are employed extensively in the manufacture of foods. Thus, there is ample exposure for sensitization to Arg-Lys-Leu-Lys (SEQ ID NO: 1) in genetically susceptible or immunocompromised individuals. Cross-reactivity does not ensure autoimmunity. The immunogenicity of the sequence is not clear but is inferred by its homology to highly immunogenic Streptococcal epitopes Ile-Arg-Leu-Arg (SEQ ID NO: 9), the cause of autoimmunity for renal glomeruli, and Gln-Lys-Ser-Lys (SEQ ID NO: 10), one target of autoimmunity for cardiac myosin.